Target-Driven Tissue-Agnostic Drug Approvals—A New Path of Drug Development

Simple Summary Precision oncology is at the forefront of personalized cancer care, utilizing tumor-agnostic therapies that target specific biomarkers, such as tumor mutational burden, microsatellite instability, mutations, or fusions, regardless of cancer type. This review aims to provide a comprehensive summary of the immune checkpoint inhibitors and the targeted therapies approved by the US Food and Drug Administration (FDA) for tumor-agnostic use. We thoroughly reviewed pembrolizumab, dostarlimab, larotrectinib, entrectinib, selpercatinib, dabrafenib plus trametinib, and trastuzumab deruxtecan across eight indications, detailing their efficacy, toxicity, and regulatory history. Additionally, we highlight the critical role of biostatistics in designing the clinical trials necessary for evaluating these treatments. These insights advance our understanding of biomarker-driven approaches, guide future research directions, and support the development of more effective, personalized cancer treatments across diverse histology. Abstract The regulatory approvals of tumor-agnostic therapies have led to the re-evaluation of the drug development process. The conventional models of drug development are histology-based. On the other hand, the tumor-agnostic drug development of a new drug (or combination) focuses on targeting a common genomic biomarker in multiple cancers, regardless of histology. The basket-like clinical trials with multiple cohorts allow clinicians to evaluate pan-cancer efficacy and toxicity. There are currently eight tumor agnostic approvals granted by the Food and Drug Administration (FDA). This includes two immune checkpoint inhibitors, and five targeted therapy agents. Pembrolizumab is an anti-programmed cell death protein-1 (PD-1) antibody that was the first FDA-approved tumor-agnostic treatment for unresectable or metastatic microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) solid tumors in 2017. It was later approved for tumor mutational burden-high (TMB-H) solid tumors, although the TMB cut-off used is still debated. Subsequently, in 2021, another anti-PD-1 antibody, dostarlimab, was also approved for dMMR solid tumors in the refractory setting. Patients with fusion-positive cancers are typically difficult to treat due to their rare prevalence and distribution. Gene rearrangements or fusions are present in a variety of tumors. Neurotrophic tyrosine kinase (NTRK) fusions are present in a range of pediatric and adult solid tumors in varying frequency. Larotrectinib and entrectinib were approved for neurotrophic tyrosine kinase (NTRK) fusion-positive cancers. Similarly, selpercatinib was approved for rearranged during transfection (RET) fusion-positive solid tumors. The FDA approved the first combination therapy of dabrafenib, a B-Raf proto-oncogene serine/threonine kinase (BRAF) inhibitor, plus trametinib, a mitogen-activated protein kinase (MEK) inhibitor for patients 6 months or older with unresectable or metastatic tumors (except colorectal cancer) carrying a BRAFV600E mutation. The most recent FDA tumor-agnostic approval is of fam-trastuzumab deruxtecan-nxki (T-Dxd) for HER2-positive solid tumors. It is important to identify and expeditiously develop drugs that have the potential to provide clinical benefit across tumor types.


Introduction
The regulatory approvals of tumor-agnostic therapies have led to the re-evaluation of the drug development process.Conventional models of drug development are histologybased.On the other hand, the tumor-agnostic drug development of a new drug (or combination) focuses on targeting a common genomic biomarker in multiple cancers, regardless of histology [1].Basket-like clinical trials with multiple cohorts allow clinicians to evaluate pan-cancer efficacy and toxicity.There are currently eight tumor-agnostic approvals granted by the FDA (Figure 1).This includes two immune checkpoint inhibitors, and five targeted therapy agents.Pembrolizumab is an anti-programmed cell death protein-1 (PD-1) antibody that was the first FDA-approved tumor-agnostic treatment for unresectable or metastatic microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) solid tumors in 2017.It was later approved for tumor mutational burden-high (TMB-H) solid tumors, although the TMB cut-off used is still debated.Subsequently, in 2021, another anti-PD-1 antibody, dostarlimab, was also approved for dMMR solid tumors in the refractory setting.Patients with fusion-positive cancers are typically difficult to treat due to their rare prevalence and distribution.Gene rearrangements or fusions are present in a variety of tumors.Neurotrophic tyrosine kinase (NTRK) fusions are present in a range of pediatric and adult solid tumors in varying frequency.Larotrectinib and entrectinib were approved for neurotrophic tyrosine kinase (NTRK) fusion-positive cancers.Similarly, selpercatinib was approved for rearranged during transfection (RET) fusion-positive solid tumors.The FDA approved the first combination therapy of dabrafenib, a B-Raf proto-oncogene serine/threonine kinase (BRAF) inhibitor, plus trametinib, a mitogen-activated protein kinase (MEK) inhibitor for patients 6 months or older with unresectable or metastatic tumors (except colorectal cancer) carrying a BRAF V600E mutation.The most recent FDA tumoragnostic approval is of fam-trastuzumab deruxtecan-nxki (T-Dxd) for HER2-positive solid tumors.It is important to identify and expeditiously develop drugs that have the potential to provide clinical benefits across tumor types.

Pembrolizumab Approved for Patients with Unresectable or Metastatic dMMR/MSI-H Cancers (23 May 2017)
The MMR system is responsible for detecting and repairing errors made during DNA replication.Mismatch repair deficiency results in a high mutational rate, which contributes to microsatellite instability.The MMR genes include mutL homolog 1 (MLH1), mutL homolog 2 (MSH2), mutL homolog 6 (MSH6), and post-meiotic segregation increased 2 (PMS2) [2].The underlying causes of microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) solid tumors are often attributed to inactivated MMR genes.This may be due to the methylation of the promoter region or epigenetic changes which may manifest as mutational alterations.This process is also predominant in hereditary cancers such as Lynch syndrome (autosomal dominant) or constitutional mismatch repair deficiency (autosomal recessive).Overview of the FDA-approved tumor-agnostic treatment.Abbreviations: Microsatellite instability-high (MSI-H); mutations/megabase (mut/Mb); deficient mismatch repair (dMMR); programmed cell death protein-1 (PD-1); programmed cell death ligand-1 (PD-L1); tumor mutational burden-high (TMB-H); B-Raf pro-to-oncogene serine/threonine kinase (BRAF); human epidermal growth factor receptor 2 (HER2); mitogen-activated protein kinase (MEK); neurotrophic tyrosine receptor kinase (NTRK); rearranged during transfection (RET).X denotes the site of the action of the drug via their mechanism of action.The MMR system is responsible for detecting and repairing errors made during DNA replication.Mismatch repair deficiency results in a high mutational rate, which contributes to microsatellite instability.The MMR genes include mutL homolog 1 (MLH1), mutL homolog 2 (MSH2), mutL homolog 6 (MSH6), and post-meiotic segregation increased 2 (PMS2) [2].The underlying causes of microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) solid tumors are often attributed to inactivated MMR genes.This may be due to the methylation of the promoter region or epigenetic changes which may manifest as mutational alterations.This process is also predominant in hereditary cancers such as Lynch syndrome (autosomal dominant) or constitutional mismatch repair deficiency (autosomal recessive).

Approved Immunotherapy (Checkpoint Inhibitors) in the
MSI-H/dMMR solid tumors may also express PD-1 biomarkers.In a study of 12,019 cancers, 75% exhibited MMR deficiency, including adenocarcinomas of the reproductive and gastrointestinal system.Among 11 tumor types, 10% of dMMR tumors were early stage, while 5% were late stage, amounting to approximately 60,000 diagnoses annually in the United States [3].Programmed cell death ligand-1 (PD-L1) blockade increased the levels of functional mutation-associated neoantigens (MANAs), suggesting that dMMR cancers with high levels of neoantigens are more susceptible to immunotherapy [4].Pembrolizumab is a monoclonal antibody targeting the PD-1 receptor present on the surface of T cells.Inhibition blocks the interaction with PD-L1/2 ligands, resulting in immunosuppression.This has broad implications for immune surveillance and DNA repair via the mismatch repair system.
Pembrolizumab was the first tissue-agnostic treatment that received accelerated FDA approval for advanced or metastatic dMMR/MSI-H solid tumors on 23 May 2017 (Table 1).The efficacy data were based on the five phase II KEYNOTE (KN) trials: KN-016, -164, -012, -028, and -158 (Figure 2).A pooled cohort of 149 patients were studied across 15 different cancer types, including 90 colorectal cancer (CRC) and 59 non-CRC cohorts (FDA, 2017 [5]).The overall objective response rate (ORR) was 39.6% (95% CI 31.7-47.9).In CRC, the ORR was 36%, whereas all other cancer types had an ORR of 46%.The duration of response was over 6 months in 78% of patients, including 7% complete response (CR) [6].The updated analysis of KN-158 evaluated 233 patients across 27 cancer types (excluding CRC).Patients were followed for a median period of 13.4 months and demonstrated an ORR of 34.3%, a median progression free survival (mPFS) of 4.1 months, and a median overall survival (mOS) of 23.5 months.The discontinuation rate due to treatment-related adverse effects (TRAEs) was 9.4% and one treatment-related death occurred due to pneumonia [7].The characteristics of the cancer (location and tumor type), pre-treatment, and other factors may contribute to the efficacy.However, more research needs to be completed to determine which specific factors contribute.The most common cancer types in the KN-158 efficacy population include malignancies of the reproductive and gastrointestinal systems.Patients with primary brain cancers did not respond to pembrolizumab [7].Consequently, the FDA included a "limitation of use" for pediatric patients with MSI-H brain tumors (FDA, 2017 [5]).Temozolomide treatment in glioblastoma patients can induce therapeutic pressure and  The most common cancer types in the KN-158 efficacy population include malignancies of the reproductive and gastrointestinal systems.Patients with primary brain cancers did not respond to pembrolizumab [7].Consequently, the FDA included a "limitation of use" for pediatric patients with MSI-H brain tumors (FDA, 2017 [5]).Temozolomide treatment in glioblastoma patients can induce therapeutic pressure and MSH-6 mutations, leading to intratumor heterogeneity and potentially reducing the immunotherapy efficacy.Recurrence may also result from treatment-induced hypermutation [13,14].Although some patients with high-grade glioma improved with treatment, not all have benefited despite the high mutation burden [15][16][17].Testing for de novo MSI-H/dMMR markers in brain tumor samples could identify treatment-responsive patients; however, their rarity in gliomas raises concerns about cost-effectiveness.KEYNOTE-177 is a phase III clinical trial that demonstrates the efficacy of 307 patients with stage IV MSI-H/dMMR CRC.Pembrolizumab monotherapy (ORR = 43.8%,mPFS = 16.5 months) had superior efficacy and survival compared to chemotherapy (ORR = 33.1%,mPFS = 8.2 months) in the first line setting [2].In contrast, other KN trials feature a heavily pretreated population.Pembrolizumab also improved the toxicity profile for this cohort (hazard ratio = 0.60, 95% CI 0.45-0.80,p = 0.0002).High-grade TRAEs (grade 3 and above) occurred in 22% and 66% of patients in the pembrolizumab and chemotherapy group, respectively.The most common high-grade adverse events were decreased neutrophil count, neutropenia, and diarrhea which resulted in a 14% treatment discontinuation rate [2].The most common TRAEs include systemic symptoms (e.g., fatigue, fever) and gastrointestinal side effects (e.g., diarrhea, nausea, abdominal pain, decreased appetite, vomiting, constipation).There were also reports of immune-mediated adverse events (1-3%) such as colitis, hepatitis, and infusion reactions.

Pembrolizumab Approved for Patients with Unresectable or Metastatic TMB-H Cancers (16 June 2020)
The tumor mutational burden (TMB) is a genomic biomarker that is defined as the number of mutations per megabase of DNA (mut/Mb).These somatic mutations may include point mutations, insertions and deletions (indels), copy number alterations (CNAs), as well as chromosomal rearrangements.The point mutations encompass synonymous (silent) and non-synonymous (missense) substitutions, the latter resulting in altered amino acid sequences and neoantigen generation.TMB-H tumors with elevated neoantigen levels often have superior responses to immunotherapy [18,19].No consensus exists on the optimal TMB-H cutoff value, leading to heterogeneous findings in the literature.Methodological variations, such as whole exome sequencing (WES) versus targeted gene panels, and decisions regarding the inclusion/exclusion of synonymous mutations, further contribute to the differences in TMB assessment.One study reviewed the TMB status of 3534 primary tumors from The Cancer Genome Atlas (TCGA) and 696 advanced tumors from Weill Cornell determined by WES [19].Fernandez and his colleagues determined that a cancer-specific threshold was superior to a pan-cancer threshold approach.In contrast, Chalmers and colleagues used a static TMB threshold of 20 mut/Mb for all cancers.Different cancers exhibit varying TMB values, likely due to differences in mutational rates.For example, prostate cancer's TMB ranges from 0.30 to 14.13 mut/Mb in TCGA, while bladder cancer ranges from 0.4 to 99.68 mut/Mb [18].Reducing the cutoff would be overly stringent, while raising it might be too lenient.Therefore, employing a cancer-specific cutoff appears to be a promising approach, especially for rare cancers.In addition to cancer type, the TMB may vary due to patient demographics, treatment intensity, and exogenous factors like smoking and UV radiation.
Pembrolizumab was approved for advanced TMB-H positive cancers on 16 June 2020, based on the phase II KEYNOTE-158 trial (Table 1).Tissue TMB-High (tTMB-H) was defined as cancers with at least 10 mut/Mb.The responses were seen across eight cancer types (Figure 2).ORR was achieved in 29.4% and 6.3% in the tTMB-H group (n = 102) and the non-tTMB-H group (n = 688), respectively [8].Despite a median TMB below the 10 muts/Mb cutoff, responders had a higher median TMB than non-responders.This implies that TMB alone may not fully predict treatment response, as individuals below the cutoff still responded, albeit with higher TMB correlating with better response rates.
Pembrolizumab has limited use for TMB-H positive central nervous system (CNS) cancers since the efficacy and safety data for this subset of patients have not been established (FDA).Patients with anal cancer also had a poor response rate.Among the 14 patients with TMB-H anal cancer, only 1 responded (ORR = 7%), whereas 9 out of 84 patients in the non-TMB-H cohort responded [10].
These findings may be attributed to several factors.For example, three patients lacked TMB information, while five patients exhibited TMB ≥ 13 mut/Mb.Given the small sample size, it is challenging to ascertain if these discrepancies stem from the specific TMB distribution in this cancer type or from data constraints.In the safety analysis of 105 patients, common TRAEs included fatigue, asthenia, hypothyroidism, and reduced appetite.Approximately 10% of patients encountered severe TRAEs.Eight patients discontinued treatment, while thirty-four patients had treatment interruptions.Fatal pneumonia related to treatment occurred in one patient with progressive disease [8].
Merck and colleagues also presented a retrospective analysis at the 2020 AACR meeting; however, the results have not been published.A pooled cohort of 2234 patients from 12 studies (KEYNOTE-001, 002, -010, -012, -028, -045, -055, -059, -061, -086, -100, 199) were analyzed using WES.TMB status was determined in 1772 patients who received pembrolizumab monotherapy and 452 patients who received chemotherapy.Among those who received pembrolizumab monotherapy, F1CDx analysis found that 433 patients (24%) had TMB ≥ 175 mut/exome which was roughly analogous to TMB ≥ 10 mut/Mb [10].The FDA also reviewed three randomized control trials for progression-free survival and overall survival among pembrolizumab monotherapy and chemotherapy cohorts as part of an exploratory post hoc analysis.The cancer types studied were non-small cell lung cancer (NSCLC), urothelial cancer, and gastric cancer from the KEYNOTE-010, KEYNOTE-045, and KEYNOTE-061 trials, respectively.
The FDA has acknowledged the inconsistencies and limitations of TMB; however, the accelerated approval of pembrolizumab for TMB-H solid tumors has been beneficial in patients who otherwise cannot receive immunotherapy.Although a TMB cutoff of ≥10 mut/Mb was used for FDA approval, Merck and colleagues also analyzed 70 patients with TMB ≥ 13 mut/Mb that demonstrated an ORR of 37% (95% CI 26-50) and 32 patients with TMB < 13 mut/Mb but ≥10 mut/Mb that demonstrated an ORR of 13% (95% CI 4-29) [10].A postmarketing study is underway in order to better understand the efficacy and clinical significance behind these different cutoffs.

Dostarlimab Approved for Patients with Unresectable or Metastatic dMMR Cancers (17 August 2021)
Dostarlimab is an IgG4-k monoclonal antibody with activity against PD-1.Dostarlimab monotherapy received accelerated FDA approval for recurrent and/or advanced dMMR cancers on 17 August 2021 (Table 1) [20].This approval was based on the phase I GARNET trial, consisting of several expansion cohorts.These included patients with dMMR/MSI-H endometrial cancer (EC, cohort A1), MMR-proficient (MMRp)/microsatellite stable (MSS) EC, NSCLC, and non-EC dMMR/MSI-H and cancers with the POLE mutation.The study also included patients with advanced, relapsed, high-grade serous, endometrioid, or clear cell ovarian, fallopian tube, or primary peritoneal cancer (PROC) without known BRCA mutation.The FDA approval was based on 209 patients across 16 different cancer histologies (Figure 2).The overall ORR was 41.6% (95% CI 34.9-48.6),including a 9.1% complete response rate and a 32.5% partial response rate.The mDOR was 34.7 months with the vast majority (>95%) of patients experiencing a duration of response for at least 6 months (FDA, 2021 [11]).In an updated analysis, 327 patients were evaluated.The most common cancer types were EC (n = 143, ORR = 45.5%) and colorectal cancer (n = 115, ORR = 43.5%).Patients also demonstrated a promising mPFS of 6.9 months (Andre et al., 2023).The most common all-grade adverse reactions were fatigue, anemia, and gastrointestinal side effects.The most common high-grade adverse reactions were anemia, fatigue, increased LFTs, sepsis, and acute kidney injury.Dostarlimab toxicity resulted in 25 treatment discontinuations and two deaths [12].
Dostarlimab has heterogenous response rates dependent on cancer histology.In the EC subgroup, the ORR is often >40%.Oaknin and colleagues demonstrated that patients with dMMR/MSI-H EC (cohort A1, n = 143) had superior efficacy compared to patients with proficient (MMRp)/MSS EC (cohort A2, n = 153).Cohort A1 and cohort A1 had a response rate of 45.5% (95% CI 37.1-54.0)and 15.4% (95% CI 10.1-22.0),respectively [12,20].In contrast, 67 patients with dMMR NSCLC had an ORR of 27% with an mDOR of 11.6 months.Patients were further characterized by PD-L1 expression.This was measured by the tumor proportion score (TPS), as follows: <1% (negative), 1-49% (low), ≥50% (high), or unknown status.The TPS status correlated with a positive trend in ORR.Those with a high TPS status (n = 5) had immune-related ORR 40%, 0% CR, 12.5% PR, and 80% DCR.Of note, those with unknown status (n = 18) had a higher ORR (44.4%) and DCR (72.2%) compared to other subgroups.There were no complete responders; however, eight patients had a partial response [21].The group with negative status had the greatest proportion of patients, which reflects the fact that a large proportion of patients with NSCLC do not have PD-L1 status.Patients with non-squamous histology also experienced superior efficacy compared to patients with squamous histology.Therefore, biomarker status and cancer subtype contribute to dostarlimab efficacy.The most commonly reported any-grade TRAEs were fatigue, hypothyroidism, and gastrointestinal symptoms (nausea, diarrhea, decreased appetite).There were eight patients (11.9%) who experienced high-grade TRAEs with fatigue being the most common.Overall, treatment was interrupted in 16 patients (23.9%) and discontinued in 4 patients (6%).The most common reasons for discontinuation were due to increased lipase (1.5%), pleural effusion (1.5%), pneumonitis (1.5%), and increased transaminases (1.5%) [21].
In patients with dMMR/MSI-H positive or POLE hypermutated non-EC (cohort F), gastrointestinal cancer compromised 93.4% of this cohort (56% colorectal cancer, 11% gastric cancer).Among 204 patients, 43.1% responded (95% CI 36.2-50.2) with a median follow-up time of 27.7 months.The median PFS was 7.1 months, although the mDOR and mOS were not reached [22].In regards to toxicity, 68.8% experienced TRAEs.Although these are only interim analyses for the different cancer subtypes, the different expansion cohorts have demonstrated the tumor-agnostic benefit of dostarlimab.Furthermore, patients with dMMR/MSI-H rectal cancer are usually poor responders to standard therapies such as chemotherapy.A recent phase II trial enrolled 16 patients with dMMR, locally advanced rectal cancer that had a minimum follow-up of 6 months.The majority of these patients had stage III rectal cancer (15 patients), whereas only 1 patient had stage II cancer.Dostarlimab reported 100% remission (95% CI 74-100) for 12 patients in June, 2022.The trial shows that 81% of patients had a resolution of their symptoms within 9 weeks of starting treatment [23].Although 75% of patients experienced any-grade adverse events (the most common being rash or dermatitis, pruritus, fatigue, and nausea), no grade 3/4 adverse events were reported.This shows promising results, especially when compared to the relatively poor response of CRC to other PD-L1 inhibitors.However, it is too early to determine if the efficacy of dostarlimab in this subset of patients can be replicated or if this is due to chance alone.The neurotrophic tropomyosin receptor kinase (NTRK) family consists of three main genes: NTRK1, NTRK2, and NTRK3.These genes code for the TRKA, TRKB, and TRKC transmembrane proteins, respectively.When neurotrophins bind to TRK receptors, dimerization of the receptor consequently occurs.This starts a series of phosphorylation events which activates the MAPK pathway (from activated TRKA) and the PI3K/AKT pathway (from activated TRKC) [24].Genetic alterations result in defective receptors leading to constitutively active kinase activity.These fusions primarily stem from chromosomal rearrangements such as deletions, inversions, and translocations.NTRK fusions, detected in less than 1% of solid tumors, have a high frequency in rare malignancies (>90%) and a low frequency in common malignancies (>5%) [25].These rare malignancies include infantile fibrosarcoma, secretory carcinomas of the breast, and salivary gland cancers.This significantly impacts patient enrollment in clinical trials and the ability to gather a sufficient number of patients for evaluating efficacy.
Only one patient out of fifty-five (2%) had brain metastasis at baseline with no intracranial response recorded [36].In this updated analysis, there were also 22 evaluable patients with baseline CNS metastasis where 68% responded to treatment (95% CI 45-86) [35].Despite the lower incidence rates of NTRK gene fusion status in patients with advanced CNS tumors, there are still unmet needs for this subset of the population.This is especially true for pediatric patients since CNS tumors are a large cause of malignancy-related mortality.Mangum and Parsons found that the intracranial ORR in pediatric patients was 38% with three complete responders and seven partial responders [37].More research is required to evaluate the true benefit of larotrectinib in these patients.
On 20 October 2023, the approval of entrectinib was expanded to include pediatric patients as young as 1 month.This was on the basis of the phase I/II STARTRK-NG trial and the phase II TAPISTRY trial.The most common cancer types studied were primary CNS tumors and infantile fibrosarcoma.Among 33 pediatric patients with NTRK fusion-positive cancers, the ORR was 70% (95% CI 51-84) with an mDOR of 25.4 months (95% CI 14.3-NE) (FDA, 2023 [29]).A safety analysis of 76 pediatric patients indicated tolerability of entrectinib, with gastrointestinal adverse events being the most common.
Unlike larotrectinib, entrectinib was designed to have more CNS penetrating efficacy.The updated integrated analysis demonstrated an intracranial ORR of 63.6% (95% CI 30.8-89.1)among 11 patients who had baseline brain metastasis [39].This is potentially due to its ability to remain in the CNS as a weak p-glycoprotein (P-gp) substrate compared to larotrectinib and crizotinib which have stronger interactions with P-gp substrates [40].P-gp has an important role at the blood-brain barrier and entrectinib's weak interaction with the efflux transporter allows for greater CNS distribution.Entrectinib can therefore meet some of the unmet needs for patients with NTRK gene fusion-positive cancers that have metastasized to the brain.
Several challenges include the heterogeneity of fusion partners, the plethora of diagnostic tools, and differing guidelines.Serious TRAEs may also not be clinically observable due to these low numbers, so more safety analyses are required to better understand these toxicities.Both larotrectinib and entrectinib are still undergoing postmarketing analysis, with the potential use of in vitro companion diagnostic devices to select patients for treatment.Although there are currently two FDA approvals targeting NTRK fusion-positive cancers, the detection and treatment of these patients can still be challenging due to the rarity of these alterations.

Dabrafenib plus Trametinib Approved for Patients with Unresectable or Metastatic BRAF V600E Positive Cancers (22 June 2022)
B-raf proto-oncogene serine/threonine kinase (BRAF) is a key enzyme in the mitogenactivated protein kinase (MAPK) signaling pathway.BRAF phosphorylates and activates downstream enzymes such as MEK and ERK.BRAF mutations (the vast majority comprising BRAF V600E ) cause the hyperproliferation of the pathway, resulting in tumor cell growth.BRAF mutations are present in melanoma (50%), NSCLC (1-5%), anaplastic thyroid carcinoma (20-50%), biliary tract cancer (5-7%), low-grade gliomas (5-15%), and glioblastomas (3%) [41].They have also been found in select cases of hairy cell leukemia, Langerhans cell histiocytosis, and CRC.The previous trials have demonstrated responses to BRAF monotherapy (such as vemurafenib); however, resistance quickly emerged.One of the known resistance mechanisms is via downstream activation ("on-target" resistance), leading to the hyperactivation of the MAPK signaling pathway regardless of BRAF inhibition.To mitigate this, MEK inhibitors were added.
Dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) were approved for unresectable or metastatic BRAF V600E positive solid tumors in adults and pediatric patients ≥ 6 years on 22 June 2022 (Table 2).However, colorectal cancers were an exception to this indication due to known resistance.The approval was based on three trials: the National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) and Rare Oncology Agnostic Research (ROAR) trials which evaluated adult patients, as well as CTMT212X2101 (X2101) which evaluated pediatric patients.This was supported by results from COMBI-d, COMBI-v, and BRF113928 (FDA, 2022, [31]).The NCI-MATCH trial was the first national study in the United States to integrate a unified method of diagnosis despite the diverse geographical locations.A total of 5954 patients with refractory malignancies were enrolled across a wide network of 1117 participating locations [42].The second trial, ROAR, was a phase II study with a specific focus on patients with rare cancers carrying the BRAF V600E mutation.Lastly, the CTMT212X2101 study (part C and part D) focused on pediatric patients with gliomas.The data for FDA approval comprised a total of 167 evaluable patients (131 adults and 36 children) across 24 unique cancer types (Figure 3).The ORR was 41% (95% CI 33-50) and 25% (95% CI 12-42) among adult and pediatric patients, respectively.Responses were durable, with 78% having a DOR 6 months or longer and 44% having a DOR 24 months or longer.The most common cancers in adults were biliary tract cancer (n = 48, ORR = 46%), high-grade glioma (n = 48, ORR = 33%), low-grade gliomas (n = 14, ORR = 50%), and various malignancies of the gastrointestinal tractThe most common TRAEs were nausea, constipation, vomiting, pyrexia, fatigue, rash, peripheral edema, headache, cough, and hemorrhage.
Corcoran and colleagues demonstrated that 43 patients with metastatic CRC had an ORR of only 12%.Responders often harbored PIK3CA co-mutations.Although the mPFS was 3.5 months, this still surpasses the survival outcomes from standard chemotherapy [43].CRC has historically been difficult to target, with many hypothesizing that this is due to "off-target" resistance from EGFR activation.Thus, encorafenib (BRAF inhibitor) plus cetuximab (EGFR inhibitor) are currently approved for these BRAF V600E -positive metastatic CRCs (FDA, 2020, [44]).There are also clinical trials assessing triple therapy.For example, dabrafenib plus trametinib plus spartalizumab exhibited a 24.3% response rate [41].However, the potential risk of increased toxicity must be evaluated in these patients.
Dabrafenib plus trametinib also received FDA approval for children with BRAF V600Epositive low-grade gliomas on 16 March 2023.This was based on the low-grade glioma sub-cohort from the TADPOLE trial (FDA, 2023 [45]) [46].Among 110 patients (73 receiving dabrafenib and trametinib therapy), the ORR was 46.6%, the mDOR was 23.7 months, and the mPFS was 20.1 months.Unfortunately, there is still no approval for BRAF V600E -positive high-grade gliomas for patients less than 6 years of age.Notably, the pooled data from the NCI-MATCH and ROAR trials demonstrated that patients with high-grade gliomas (n = 48) had the lowest response rate (ORR = 33%) as well as an mDOR of 3.9 months.However, a post hoc analysis found that stratifying patients by age (18-39 years) improved the objective response rate to 50%, similar to the low-grade glioma cohort [41].Although only 5-10% of pediatric high-grade gliomas harbor a BRAF V600E mutation, there is a high mortality associated with these malignancies.This represents the clinical benefit of these medications among young adults compared to the pediatric population.The rearranged during transfection (RET) gene encodes proto-oncogenic transmembrane receptor tyrosine kinases (RTKs).They are activated by binding to the glial cell-linederived neurotrophic factor (GDNF) ligands.This forms a heterodimeric ligand-co-receptor complex, which initiates signaling cascades crucial for cellular growth and differentiation.This pathogenesis is believed to arise from chromosomal instability, leading to gene fusions with partners such as KIF5B.RET gain-of-function mutations are implicated in malignancies such as invasive breast cancers (30-70%), pancreatic ductal adenocarcinomas (50-60%), sporadic papillary thyroid carcinomas (2.5-73%), and NSCLC (1-3%) [47].They have also been reported in Hirschsprung's disease (loss-of-function mutations) and MEN2 syndrome (inherited form).RET fusions have a frequency of <1% across multiple cancers.They are prevalent in rare cancers such as MASC, while they are rare in common cancers.
Selpercatinib [LOXO-292/LY3527723], a potent RET inhibitor with CNS-penetrating activity, gained orphan drug status due to the lack of effective targeted therapies [48].On 21 September 2022, selpercatinib was granted FDA approval for RET fusion-positive advanced solid tumors in adult and pediatric patients (Table 2).This was based on the ongoing phase I/II trial LIBRETTO-001, spanning 30 sites across eight countries [49].Patients weighing <50 kg received 120 mg and those ≥ 50 kg received 160 mg tablets, taken twice daily for a maximum of 2 years.The efficacy population consisted of 41 patients across 14 unique cancer types, excluding NSCLC and thyroid cancer (Figure 3).Pancreatic adenocarcinoma (27%), CRC (24%), salivary cancer (10%), and cancers of unknown primary (7%) were the most common.The response rate was 43.9% (95% CI 28.5-60.3),including two complete responders and sixteen partial responders.Patients had an mPFS of 13.2 months, an mOS of 18 months, as well as an mDOR of 24.5 months.The majority of patients had a response for at least 6 months (>65%) (FDA, 2022 [32]).The safety population included 796 patients.The most commonly reported toxicities were diarrhea, constipation, nausea, fatigue, dry mouth, hypertension, rash, and headache.The most common serious TRAEs were elevated hypertension in 22% of patients, as well as elevated ALT and AST in 16% and 13% of patients, respectively [49].
After 16 months of further follow-up, selpercatinib continued to have durable responses in RET fusion-positive gastrointestinal cancers.Patients with pancreatic cancer (n = 13) had a response rate of 53.8% (95% CI 25.1-80.8),including one complete responder and six partial responders.Responses were durable with an mDOR > 50 months and mPFS was 5.6 months.Patients with colorectal cancer (n = 13) had an ORR of 30.8% (95% CI 9.1-61.4),an mDOR of 13.3 months, as well as an mPFS of 9.1 months [50].CRC and pancreatic cancer face efficacy challenges due to resistance.RET fusions are also rare in both cancer types, posing diagnostic challenges.A significant number of CRC patients also had MSI-H status, suggesting that some may benefit from combinational therapies.This underlines the importance of genomic testing and their role in treatment.
The FDA reported the most common (≥20%) TRAEs involved cytopenia of three cell lines (leukocytes, erythrocytes, and platelets), gastrointestinal side effects, as well as decreased electrolytes (FDA, 2024 [33]).In DESTINY-PanTumor02, 58.7% experienced grade 3 TRAEs.This also contributed to treatment discontinuation in 11.6% of patients [34,56].Three deaths were attributed to interstitial lung disease.Notably, patients with a past medical history of interstitial lung disease and/or pneumonitis (either requiring steroid treatment or a diagnosis of significant cardiac disease at screening) were excluded.Similarly, patients with lung cancers had an increased risk of treatment-related ILD.The NSCLC cohort had a significant ILD rate of 26%.Patients who received the FDA-recommended dose (5.4 mg/kg) and those who had a higher dose (6.4 mg/kg) had a treatment-related ILD rate of 20% and 5%, respectively [58].This warrants careful monitoring and screening for pulmonary injury.Early detection and management is vital to preventing fatal respiratory toxicity.
In DESTINY-PanTumor02, patients with IHC 3+ (HER2 positive) cancers had a more favorable objective response compared to IHC 2+ (borderline) cancers.The pan-cancer ORR was 61.3% versus 27.2%, respectively [34,56].The options for cancers with lower HER2 expression have limited treatment options.However, a subset of patients with HER2 negative cancers may still benefit from T-DXd.Currently, diagnostic guidelines and recommendations have been established in HER2-positive and HER2-low breast cancer.However, this has not been established for other cancer types.T-DXd is the first ADC to be granted agnostic approval for HER2 IHC3+ cancers.T-DXd's approval for HER2 IHC3+ cancers mark the first ADC to receive agnostic approval, potentially opening avenues for future ADC agnostic approvals.Accurate pan-cancer diagnostic recommendations are essential for identifying the patients most likely to respond.

Biostatistics-Trial Design and Conducting in Tissue-Agnostic Drug Approvals
Biostatistics plays a role in designing and conducting clinical trials and has had a significant impact on cancer research and new drug developments.Traditional oncology clinical trials are designed to estimate the average effect, often derived from randomized clinical trials of unselected patients with all-comers.This has been the mainstay of drug approvals for decades.Recent tissue-agnostic clinical trials that utilize molecular genetics and biomarker-driven strategies have made a significant paradigm shift in precision cancer medicine (Figures 2 and 3).As a result, biomarker-driven, tissue-agnostic indications have seen approval by the US FDA (Figure 1).This paradigm shift raises some issues regarding biostatistical inference and study design.Berry [60] stated, "it is ironic that we take the same clinical trial approach to evaluate all manner of potentially amazing transformative experimental therapies and yet we don't experiment with the design of the clinical trial itself".There have been remarkable advances in the development of molecularly targeted agents.However, the modernization of clinical trial designs has been outpaced.Now, alternative designs have emerged to catch up to these advances.There are two main approaches for trial design.One approach is an all-comers design that treats all patients with the specific disease and determines the indication through subgroup analysis.The other approach is an enrichment design that enrolls limited patients with the therapy's targeted genomic or molecular aberrations.Some novel enrichment design/adaptive biomarker-driven trials have been developed such as master protocol, basket trial, umbrella trial, and platform trial.These novel designs investigate hypotheses through concurrent multiple treatment arms or populations, allowing for the addition or removal of arms during the trials [61].A number of authors have noted confusion regarding the definitions of these terms [62][63][64][65], and some authors have offered a taxonomy of trial designs [60,66,67].Park et al. [61] performed a systematic review of basket trials, umbrella trials, and platform trials [61].They identified 49 basket, 18 umbrella, and 16 platform trials, and the median sample sizes of the basket and umbrella trials were 205 and 346, respectively.The majority of basket trials were exploratory non-randomized trials, while most umbrella trials were also exploratory but with more use of randomization than basket trials.Notably, basket trials emerged as a strong tool for evaluating biomarker-targeted therapies among multiple tumor histologies.Many of the basket trials were designed to estimate high and durable objective responses and were conventionally conducted within the phase I and phase II setting [61,68].Only a few phase III studies, ALCHEMIST (NCT02193282, NCT02595944, and NCT02201992) and CLUSTER (NCT02059291), employed a basket design.Despite the emergence of novel trial designs, there are remaining challenges for designing and performing clinical trials.In 2022, the FDA released a guidance document describing "Tissue Agnostic Drug Development in Oncology".Adequate justification for the number of subjects and cancer types in each trial with a meaningful treatment effect for a tissueagnostic oncology drug development is required while controlling for Type I error [69].With basket trials, basket-wise and family-wise type I errors arise [63,70].The basketwise type I error rate is for an individual tumor type, while the family-wise type I error rate is a multiplicity adjusted error rate that represents the false positive rate for at least one of the null baskets.Despite the burgeoning number of potential anticancer drugs and increased numbers of people with cancer, fewer clinical investigations are possible because of limited resources [2].With traditional statistical approaches, it is a challenge to have an adequately powered sample size with strong control of type I error rate for each individual tumor type due to the limited resources.It is becoming common for trials to be tailored to detect enhanced efficacy in a patient subpopulation, and large clinical trials will soon be less possible, if not impossible [71,72].Several authors such as Menis et al. [63] and Burock et al. [73] have stated that the goal of cancer clinical trials in this era of precision medicine should be to conduct 'trials designed to learn' which lead to 'trials designed to conclude', which begins with identifying large and meaningful differences within small, molecularly enriched groups of patients, often referred to as 'home runs' [74].With advances in biology, every cancer will become a rare cancer.Statistical approaches will have to evolve to make the best use of limited resources through the use of novel designs.Before the era of these novel designs, the majority of statistical analyses investigated population/marginal-level responses of intervention.In basket trials, the marginal response rate from pooled analysis may increase the statistical power because of the overall sample size.However, heterogeneity and imbalanced enrollment are common among basket trials, and pooled analysis risks erroneous conclusions as outcomes from over-represented tumor types may be extrapolated to individual histology.Basketindependent analysis could address this issue by avoiding the assumption of global effect, but it is limited by sample size and accrual, and one may need to adopt an analysis strategy that controls family-wise type I errors among multiple hypothesis tests [68].In addition, the importance of bioinformatics, now more than ever, cannot be overstated as molecular profiling of tumor biopsies plays an increasingly important role in cancer research [75].Appropriate bioinformatic methods for managing and integrating large and complex data are necessary to make possible the statistical analysis needed to discover and validate predictive models based on omics technologies [72].

Future Directions and Challenges
Examples of agents that are showing promising clinical benefit across tumor types but have not yet received regulatory approval are FGFR inhibitors and KRAS G12C inhibitors.

Targeting Fibroblast Growth Factor Receptor (FGFR)
Fibroblast growth factor receptor (FGFR) is a family of transmembrane receptor tyrosine kinases.FGFR1 (3.5%), FGFR2 (1.5%), FGFR3 (2%), and FGFR4 (0.5%) alterations are present in a wide range of histologies.These include cancers of the breast, lung, and gastrointestinal tract [76].Erdafitinib is an anti-FGFR 1-3 agent that is being evaluated in the ongoing phase II RAGNAR trial.Among 217 patients with 16 unique cancer types, the ORR was 30% after 17.9 months of follow-up.Erdafitinib showed a survival benefit with mPFS of 4.2 months and mOS of 10.7 months.Approximately 70% of patients experienced grade 3 adverse events, with >8% of these being serious TRAEs.However, no treatment-related deaths occurred [77,78].Only patients aged 12 or older were enrolled; therefore, efficacy in younger patients is unestablished.FGFR alterations had an incidence of 3% of solid tumors across 19 different histologies including rhabdomyosarcomas and gliomas [79].These cancer types are more prevalent in pediatric patients.The role of pan-FGFR inhibitors in this cohort as well as the general tumor-agnostic avenue requires a larger patient population.

Tackling Kirsten Rat Sarcoma Virus (KRAS G12C )
Kirsten rat sarcoma virus (KRAS) is a gene that encodes for the K-Ras protein, a crucial aspect of the MAPK signaling pathway responsible for cell survival and apoptosis.It is frequently mutated in CRC (45%), pancreatic cancers (90%), and NSCLC (35%) [80].KRAS has historically been difficult to target due to its complex biological structure and GDP-GTP bound state.The emergence of KRAS G12C inhibitors, sotorasib and adagrasib, has garnered interest since their approval for NSCLC.Despite their success, there are still many patients with KRAS-positive solid tumors that do not have satisfactory therapies.Divarasib (GDC-6036) is a more selective and potent KRAS G12C inhibitor that has demonstrated improved efficacy.In a recent analysis of a phase 1 trial, 137 patients receiving divarasib were evaluated.The results were promising, revealing a 53.4% confirmed response rate in 60 patients with NSCLC, and a median progression-free survival of 13.1 months.In 55 patients with colorectal cancer, a 29.1% confirmed response rate and a median progression-free survival of 5.6 months were observed.The most common TRAEs were gastrointestinal toxicity, including nausea (74%), diarrhea (61%), and vomiting (58%).Of those who experienced adverse events, four patients discontinued treatment and nineteen required a dose reduction [81].Only around 13% of solid tumors harbor a KRAS G12C mutation [80].There are still unmet needs for patients who harbor alterations of other KRAS isoforms, such as KRAS G12D .Pan-KRAS inhibitors are also currently in development.

Conclusions
The ability to develop agents for tumor-agnostic indications holds tremendous promise, especially in rare cancers, where it may not be feasible to conduct adequately powered trials.Increasing the utilization of next generation sequencing is identifying the presence of molecular targets in pediatric and adult cancers, allowing enrollment in basket trials.The challenges around trial enrollment, incomplete understanding of the biology of target in the context of underlying histology, development of resistance, tumor heterogeneity, and management of toxicity, remain to be addressed.

Figure 2 .
Figure 2. Timeline of FDA-approved immune checkpoint inhibitors for tissue-agnostic indications.

Figure 2 .
Figure 2. Timeline of FDA-approved immune checkpoint inhibitors for tissue-agnostic indications.

3 .
Approved Targeted Cancer Therapeutics in the Subject of Histology-Agnostic Drug Development 3.1.Larotrectinib Approved for Patients with Unresectable or Metastatic NTRK Gene Fusion-Positive Cancers (26 November 2018)

Figure 3 .
Figure 3. Timeline of FDA-approved targeted therapy for tissue-agnostic indications.Figure 3. Timeline of FDA-approved targeted therapy for tissue-agnostic indications.

Figure 3 .
Figure 3. Timeline of FDA-approved targeted therapy for tissue-agnostic indications.Figure 3. Timeline of FDA-approved targeted therapy for tissue-agnostic indications.

Table 1 .
Summary of FDA-approved immunotherapy.

Table 2 .
Summary of FDA-approved targeted therapy.